All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art.
Plants have two phases to their life cycle: the diploid phase, or sporophyte, that ends in meiosis to produce haploid cells, and the haploid phase, or gametophyte, in which mitotic proliferation to produce a haploid plant includes differentiation of a subset of cells as gametes prepared for fertilization to reconstitute a diploid organism. Both the egg-producing haploid plant, the megagametophyte, and the sperm-producing haploid plant, the microgametophyte, are genetically active, hence gametophyte phenotype reflects haploid allele type. In contrast, gamete properties in animals are determined almost entirely by gene expression in progenitor diploid cells. Flowering plants are further distinguished from animals by the process of double fertilization. Two cells of the female gametophyte, the egg and central cell, are fertilized by two typically genetically identical sperm cells of the male gametophyte to produce the embryo and endosperm, respectively, of the seed.
In angiosperms the polygonum type of megasporogenesis is most common, occurring in 70% of species, including Arabidopsis and maize. In these plants the chalazal megaspore, one of the meiotic products of the megaspore mother cell, undergoes three rounds of free nuclear division followed by cellularization to give rise to a seven-celled embryo sac (FIG. 1). The free nuclear divisions are invariant in number, tightly regulated as indicated by their synchrony, and accompanied by stereotypical nuclear migrations. The angiosperm female gametophyte, called the embryo sac, consists of four cell types: synergids, antipodals, egg, and central cell (Drews et al., 1998; Grossniklaus and Schneitz, 1998; Yang and Sundaresan, 2000).
Despite the limited size of the gametophytes in flowering plants a very large number of genes are essential for haploid development. The gametophytes undergo mitosis, cell growth, and organelle biogenesis. Cells exchange signals for differentiation and for interaction with the surrounding diploid tissues. Gametophytes acquire attributes important in self vs. non-self recognition during pollination, and gametes acquire factors required for successful fertilization. Many basic cellular processes are required in gametophytes (e.g. tip growth of cells in the pollen tube of the microgametophyte; gamete fusion; cell-cell attraction; mitosis; cytokinesis; intracellular trafficking; cell death).
Demonstration that the entire genome, rather than specific chromosomes or a few chromosomal segments, are important comes from classical cytogenetic analyses in maize. In the ˜3000 cM maize genetic map, there are only a few regions in which short deletions still permit production of viable gametes, i.e. 2 cM deletions from anther ear1 to bronze2 on chromosome 1 and from shrunken1 to bronze1 on chromosome 9 (Patterson, E. B. 1978). More convincingly Patterson and others exploited more than 850 reciprocal translocation stocks, representing 1700 deficiencies, to establish that all caused pollen abortion and about 90% resulted in megagametophyte lethality (Coe et al., 1988). Presumably some nutritional defects lethal to pollen, which is sealed from metabolic exchange with the surrounding diploid tissues for days, can be compensated for in megagametophytes, which continue to absorb nutrients from their diploid mother.
Gametophyte mutations result in characteristic phenotypes and modes of transmission. Heterozygotes for female gametophyte mutations are expected to have reduced fertility, because half of the embryo sacs inherit the mutant allele. Male gametophyte mutations do not cause reduced seed set because there is normally excess pollen. However, for both male and female gametophyte mutations the mutant allele is found at a reduced frequency in progeny when the affected gamete is involved in the cross. Because these mutations act after meiosis they are transmitted poorly or not at all, as are loci linked to them.
Mutations that act in the gametophyte generation have been identified recently in several species by screening for poor transmission through the gametes and for semisterility of mutant heterozygotes (Feldmann et al., 1997; Moore et al., 1997; Howden et al., 1998; Christensen et al., 1998; Christensen et al., 2002; Shimizu and Okada, 2000). The mutants fall into several categories: gametophytes that arrest early in development; well developed, but morphologically aberrant gametophytes and morphologically normal gametophytes that nevertheless fail to function. All developmental steps depicted in FIG. 1 are represented by at least one mutant. The largest class of mutants contains those that arrest development early. Although some of these mutations may be in genes with specific roles during embryo sac development, many of them likely are required for functions in all cell types. One example of this is PROLIFERA of Arabidopsis (Springer et al., 1995). PRO shows homology to MCM2-3-5 genes required for DNA replication and cell cycle control. The mutant phenotype, sequence, and expression pattern of PRO suggest it is required in all dividing cells.